Various bacteria are known to secrete proteases at some stage in their life cycles. Bacillus species produce two major extracellular proteases, a neutral protease (a metalloprotease inhibited by EDTA) and an alkaline protease (or subtilisin, a serine endoprotease). Both generally are produced in greatest quantity after the exponential growth phase, when the culture enters stationary phase and begins the process of sporulation. The physiological role of these two proteases is not clear. They have been postulated to play a role in sporulation (J. Hoch, (1976) Adv. Genet. 18, 69-98; P. Piggot, et al., (1976) Bact. Rev. 40, 908-962; and F. Priest, (1977) Bact. Rev. 41, 711-753), to be involved in the regulation of cell wall turnover (L. Jolliffe, et al., (1980) J. Bact. 141, 1199-1208), and to be scavenger enzymes (Priest, Id.). The regulation of expression of the protease genes is complex. They appear to be coordinately regulated in concert with sporulation, since mutants blocked in the early stages of sporulation exhibit reduced levels of both the alkaline and neutral protease. Additionally, a number of pleiotropic mutations exist which affect the level of expression of proteases and other secreted gene products, such as amylase and levansucrase (Priest, Id.).
Subtilisin has found considerable utility in industrial and commercial applications (see U.S. Pat. No. 3,623,957 and J. Millet, (1970) J. Appl. Bact. 33, 207). For example, subtilisins and other proteases are commonly used in detergents to enable removal of protein-based stains. They also are used in food processing to accommodate the proteinaceous substances present in the food preparations to their desired impact on the composition.
Classical mutagenesis of bacteria with agents such as radiation or chemicals has produced a plethora of mutant strains exhibiting different properties with respect to the growth phase at which protease excretion occurs as well as the timing and activity levels of excreted protease. These strains, however, do not approach the ultimate potential of the organisms because the mutagenic process is essentially random, with tedious selection and screening required to identify organisms which even approach the desired characteristics. Further, these mutants are capable of reversion to the parent or wild-type strain. In such event the desirable property is lost. The probability of reversion is unknown when dealing with random mutagenesis since the type and site of mutation is unknown or poorly characterized. This introduces considerable uncertainty into the industrial process which is based on the enzyme-synthesizing bacterium. Finally, classical mutagenesis frequently couples a desirable phenotype, e.g., low protease levels, with an undesirable character such as excessive premature cell lysis.
Special problems exist with respect to the proteases which are excreted by Bacillus. For one thing, since at least two such proteases exist, screening for the loss of only one is difficult. Additionally, the large number of pleiotropic mutations affecting both sporulation and protease production make the isolation of true protease mutations difficult.
Temperature sensitive mutants of the neutral protease gene have been obtained by conventional mutagenic techniques, and were used to map the position of the regulatory and structural gene in the Bacillus subtilis chromosome (H. Uehara, et al., (1979) J. Bact. 139, 583-590). Additionally, a presumed nonsense mutation of the alkaline protease gene has been reported (C. Roitsch, et al., (1983) J. Bact. 155, 145-152).
Bacillus temperature sensitive mutants have been isolated that produce inactive serine protease or greatly reduced levels of serine protease. These mutants, however, are asporogenous and show a reversion frequency to the wild-type of about from 10.sup.-7 to 10.sup.-8 (F. Priest, Id. p. 719). These mutants are unsatisfactory for the recombinant production of heterologous proteins because asporogenous mutants tend to lyse during earlier stages of their growth cycle in minimal medium when compared to sporogenic mutants, thereby prematurely releasing cellular contents (including intracellular proteases) into the culture supernatant. The possibility of reversion also is undesirable since wild-type revertants will contaminate the culture supernatant with excreted proteases.
Bacillus sp. have been proposed for the expression of heterologous proteins, but the presence of excreted proteases and the potential resulting hydrolysis of the desired product has retarded the commercial acceptance of Bacillus as a host for the expression of heterologous proteins. Bacillus megaterium mutants have been disclosed that are capable of sporulation and which do not express a sporulation-associated protease during growth phases. However, the assay employed did not exclude the presence of other proteases, and the protease in question is expressed during the sporulation phase (C. Loshon, et al., (1982) J. Bact. 150, 303-311). This, of course, is the point at which heterologous protein would have accumulated in the culture and be vulnerable.
Accordingly, an object of U.S. patent application Ser. No. 614,615 (EPO Publication No. 0130756) is the construction of a Bacillus strain which is substantially free of extracellular neutral and alkaline protease during all phases of its growth cycle and which exhibits substantially normal sporulation characteristics. The need disclosed therein is for a non-revertible, otherwise normal protease deficient organism that can be transformed with high copy number plasmids for the expression of heterologous or homologous proteins.
The present inventors have discovered that certain mutant subtilisins (made according to the methods disclosed in EPO Publication No. 0130756) were not completely secreted from Bacillus expression hosts which were rendered incapable of expressing and secreting enzymatically active neutral protease and subtilisin. These mutant subtilisins, containing mutations within the active site of subtilisin, were found to be incapable of autoproteolytic maturation and thus were bound to the Bacillus cell membrane making them far more difficult to isolate than if they were completely secreted into the culture medium. The inventors discovered that such mutants can be released from the surface of such Bacillus expression hosts by contacting the host cells with an enzymatically active subtilisin.
Accordingly, an object of the invention herein is to provide processes for producing a heterologous non-human carbonyl hydrolase which is not secreted but which is bound to the surface of an expression host which does not produce extracellular enzymatically active subtilisin.
In addition, an object of the invention is to provide processes for producing heterologous non-human carbonyl hydrolases which are released from the surface of a host cell by an enzymatically active subtilisin produced by the host cell.
A further object of the present invention is to provide processes for preparing heterologous polypeptides from a fusion polypeptide which can be cleaved to produce a desired heterologous polypeptide.
Still further an object of the present invention is to provide non-human carbonyl hydrolases which are normally membrane-associated and not released from the host cell expressing the hydrolase. Such hydrolases are substantially free of the host cell membrane with which they are normally associated.